Culture conditions: 32^◦C, 5% CO₂

Origin

Mouse keratinocytes from C57Bl6 strain.

Substrate

Keratinocytes grow optimally on feeder cells (e.g. 3T3J2 fibroblasts) or on collagen I-coated surfaces.

Thawing and plating cells

• Thaw 1 freezing vial containing 2×10⁶ cells (37°C water bath).
• Transfer cells into 15 ml plastic tube containing 9 ml of pre-warmed medium and spin for 5 min at 1.000 rpm (at room temperature).
• Re-suspend in 3 ml of FDA medium and plate on collagen-coated (5 μg/cm²) culture dish (3 cm diameter).
• Change medium next day.

Subculture

• To maintain their proliferative potential, Ca²⁺ must be depleted to 50 μM in the serum and must be absent from all solutions (e.g. PBS, DMEM etc…).
• Passage cells only when they are confluent (density 5 to 6×10⁴ cells/cm²).
• Typical dilution is 1:2 (1:3 is the maximum).
• Trypsinize cells with 0.1% trypsin/0.02% EDTA (5 min at 37°C) until cells detach.
• Pellet the cells (1000 rpm for 4 minutes, RT).
• Re-suspend cells in fresh medium and re-plate onto 2 dishes.
• Replace medium every 2-3 days.

Freezing

2×10⁶ cells/mL per vial (90 % FBS low Ca²⁺ and 10% DMSO).

Induction of terminal differentiation by addition of Ca²⁺

• The next day after cells were seeded, change the medium from normal FAD media to FAD+ Medium containing 1.2 mM CaCl₂.
• Experiments should be performed 3 days after plating and 48-50h after the calcium shift.

NB: Experiments in low calcium conditions can be performed on the second day after plating.

Culture of keratinocyte Feeder cells

Origin

• MEFs (3T3J2) fibroblasts.
• SNLP (3T3J2) fibroblasts.

Subculture

• Feeder cells grow in FDA medium, without insulin, cholera toxin and hydrocortisol.
• Typical passage 1:6 (1:10 is maximum). Use 0.05% Trypsin/EDTA.
• Before using them as feeders, cells must be inactivated by mitomycin C-treatment.

Mitomycin C-treatment

1. Add 10 μg/ml mitomycin C to a confluent culture of feeder cells and place in incubator for 2-3 h.
2. Wash dishes 5 times with PBS and 1 time with medium.
3. Freeze cells (10⁶ cells/ml).
4. Before using them, test they no longer divide for 4-5 days. Only use after this test has been performed successfully.

​Keratinocytes feeding

• Plate feeders (50.000 cells /cm² = confluence) on collagen I-coated dishes 2h before plating keratinocytes, or together.

from Thomas Magin posted by Oualid Haddad 2013